PCR based molecular characterization of Nepenthes khasiana Hook. f.—pitcher plant

  • B. S. BhauEmail author
  • K. Medhi
  • T. Sarkar
  • S. P. Saikia
Research Article


Nepenthes khasiana Hook. f. belonging to monotypic family Nepenthaceae is a rare, endangered, dioecious member of the carnivorous plant found in North-East India. The plant is endemic to the Indian state of Meghalaya and is distributed throughout the state from West Khasi hills to East Khasi hills, Jaintia hills and East, West to South Garo hills from 1,000 to ca. 1,500 m altitude. Multi-locus analysis using PCR based Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) markers were used for the first time to assess the genetic diversities of N. khasiana Hook. f. collected from different parts of Meghalaya. It was observed that RAPD analysis showed more polymorphism than ISSR fingerprinting in revealing genetic polymorphism in N. khasiana Hook. f. The result of cluster analysis by using UPGMA method showed that the groups based on pooled RAPD–ISSR genetic similarity were more similar than the groups based on RAPD. Furthermore, genetic similarity reveals variability within the population at Jarain of Jaintia hills, while between populations the Baghmara region differs from the others with at least 40% dissimilarity. The results show a broad range of genetic diversity within the populations of N. khasiana Hook. f.


Conservation Genetic diversity ISSR Molecular markers Nepenthes khasiana Hook. f. Pitcher plant Polymorphism RAPD 



Authors are grateful to Dr. P. G. Rao, Director NEIST for providing facilities and constant encouragement to undertake this work.


  1. Avise JC (1994) Mitochondrial DNA polymorphism and a connection between genetics and demography of relevance to conservation. Conserv Biol 9:686–690. doi: 10.1046/j.1523-1739.1995.09030686.x CrossRefGoogle Scholar
  2. Bramwell D (1972) In: Valentine DH (ed) Endemism in the flora of the Canary Islands. Taxonomy, phytogeography and evolution. Academic Press, London, pp 141–159Google Scholar
  3. Cannon JR, Lojanapiwanta V, Raston CL, Sinchai W, White AH (1980) The quinones of Nepenthes rafflesiana. Aust J Chem 33:1075–1093Google Scholar
  4. Chaveerach A, Tanomtang A, Sudmood R, Tanee T (2006) Genetic diversity among geographically distributed population of Nepenthes mirabilis. Biologia (Bratisl) 61:295–298. doi: 10.2478/s11756-006-0054-4 CrossRefGoogle Scholar
  5. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  6. Eguiarte LE, Pinero D (1990) Genética de la conservación: leones vemos, genes no sabemos. Ciencias 4:34–47Google Scholar
  7. Esselman EJ, Jianqiang L, Crawford DJ, Windus JL, Wolfe AD (1999) Clonal diversity in the rare Calamagrostis porteri ssp. insperata (Poaceae): comparative results for allozymes and random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers. Mol Ecol 8:443–451. doi: 10.1046/j.1365-294X.1999.00585.x CrossRefGoogle Scholar
  8. Fang DQ, Roose ML (1997) Identification of closely related citrus cultivars with inter-simple sequence repeat markers. Theor Appl Genet 95:408–417. doi: 10.1007/s001220050577 CrossRefGoogle Scholar
  9. Fracaro F, Zacaria J, Echeverrigaray S (2005) RAPD based genetic relationships between populations of three chemotypes of Cunila galoioides Benth. Biochem Syst Ecol 33:409–417. doi: 10.1016/j.bse.2004.10.017 CrossRefGoogle Scholar
  10. Frankel HO, Soulé ME (1981) Conservation and evolution Cambridge. Cambridge University Press, CambridgeGoogle Scholar
  11. Gallie DR, Chang SC (1997) Signal transduction in the carnivorous plants Sarracenia purpurea. Plant Physiol 115:1461–1471. doi: 10.1104/pp.115.4.1461 CrossRefPubMedGoogle Scholar
  12. Gary AJ (1987) Genetic change during succession in plants. In: Gary AJ, Edwards PJ, McCauley J (eds) Colonization, succession and stability. Blackwell Scientifc Publications, OxfordGoogle Scholar
  13. Gitzendanner MA, Soltis PS (2000) Patterns of genetic variation in rare and widespread plant congeners. Am J Bot 87:783–792. doi: 10.2307/2656886 CrossRefPubMedGoogle Scholar
  14. Givnish TJ, Burkhardt EL, Happel RE, Weintraub JW (1984) Carnivory in the bromeliad Brocchinia reducta, with a cost/benefit model for the general restriction of carnivorous plants to sunny, moist, nutrient poor habitats. Am Nat 124:479–497. doi: 10.1086/284289 CrossRefGoogle Scholar
  15. González-Astorga J, Castillo-Campos G (2004) Genetic variability of narrow endemic tree Antirhea aromatica Castilo-Campos and Lorence (Rubiaceae, Guettardeae) in a tropical forest of Mexico. Ann Bot (Lond) 93:521–528. doi: 10.1093/aob/mch070 CrossRefGoogle Scholar
  16. González-Astorga J, Cruz-Angón A, Flores-Palacios A, Vovides AP (2004) Diversity and genetic structure of the Mexican endemic epiphyte: Tillandsia achyrostachys E. Morr. ex Baker var. achyrostachys (Bromeliaceae). Ann Bot (Lond) 94:545–551. doi: 10.1093/aob/mch171 CrossRefGoogle Scholar
  17. Harbone JB (1982) Introduction to ecological biochemistry. Academic Press, New YorkGoogle Scholar
  18. Heubl G, Bringmann G, Meimberg H (2006) Molecular phylogeny and character evolution of carnivorous plant families in caryophyllales. Plant Biol 8:821–830. doi: 10.1055/s-2006-924460 CrossRefPubMedGoogle Scholar
  19. Hoebee SE, Young AG (2001) Low neighbourhood size and high interpopulation differentiation in the endangered shrub Grevillea iaspicula McGill (Proteaceae). Heredity 86:489–496. doi: 10.1046/j.1365-2540.2001.00857.x CrossRefPubMedGoogle Scholar
  20. Holsinger KE, Gottlieb LD (1991) Conservation of rare and endangered plants: principles and prospects. In: Falk DA, Holsinger KE (eds) Genetics and conservation of rare plants. Oxford University Press, New York, pp 195–208Google Scholar
  21. Hooker JD (1886) The flora of British India. vol 5. pp 68–71Google Scholar
  22. Jain SK, Baishya AK (1977) Nepenthes khasiana: an endangered species. Hornbill: 17–18Google Scholar
  23. Jain SK, Sastri ARK (1980) Threatened plants of India- a state-of-the-art report Bot Survy India, Calcutta 48Google Scholar
  24. Jayaram K, Prasad MNV (2005) Rapidly in vitro multiplied Drosera as reliable source for plumbagin bioprospection. Curr Sci 89:447–448Google Scholar
  25. Joseph J, Joseph K (1986) Insectivorous plants of khasi and Jaintia hills, Meghalaya, India. Botanical Survey of India, CalcuttaGoogle Scholar
  26. Kanjilal UN, Kanjilal PC, De RN, Das A (1940) Nepentheses. Flora of Assam, vol IV, pp 25Google Scholar
  27. Kharkongar P, Joseph J (1981) Folklore medico botany of rural Khasi and Jantia tribes in Meghalaya. In: Jain SK (ed) Glimpses of Indian Ethnobotany. Oxford IBH Publishing Co., New Delhi, pp 124–136 Google Scholar
  28. Khoshbakht K, Hammer K (2007) Threatened and rare ornamental plants. J Agric Rural Dev Trop Subtrop 108:19–39Google Scholar
  29. Kumar YK, Haridasan S, Rao RR (1980) Ethnobotanical notes on certain medicinal plants among some Garo people around Balphakram Sanctury in Meghalaya. Bull Bot Surv India 22:161–165Google Scholar
  30. Kurata K, Jaffré T, Setoguchi H (2008) Genetic diversity and geographical structure of the pitcher plant Nepenthes vieillardii in New Caledonia: a chloroplast DNA haplotype analysis. Am J Bot 95:1632–1644. doi: 10.3732/ajb.0800129 CrossRefGoogle Scholar
  31. Lewontin RC, Hubby JL (1966) A molecular approach to the study of genetic heterozygosity in natural populations. II. Amount of variation and degree of heterozygosity in natural populations of Drosophila pseudoobscura. Genetics 54:595–609PubMedGoogle Scholar
  32. Li JM, Jin ZX (2006) High genetic differentiation revealed by RAPD analysis of narrowly endemic Sinocalycanthus chinensis Cheng et S. Y. Chang, an endangered species of China. Biochem Syst Ecol 34:725–735. doi: 10.1016/j.bse.2006.06.010 CrossRefGoogle Scholar
  33. Lim SH, Phua DCY, Tan HTW (2000) Primer design and optimization for RAPD analysis of Nepenthes. Biol Plant 43:153–155. doi: 10.1023/A:1026535920714 CrossRefGoogle Scholar
  34. Loveless MD, Hamrick JL (1984) Ecological determinants of genetic structure in plant population. Annu Rev Ecol Syst 15:65–95. doi: 10.1146/ CrossRefGoogle Scholar
  35. Maki M, Asada Y (1998) High genetic variability revealed by allozymic loci in the narrow endemic fern Polystichum otomasui (Dryoperidaceae). Heredity 80:604–610. doi: 10.1046/j.1365-2540.1998.00328.x CrossRefGoogle Scholar
  36. Mantel NA (1967) The detection of disease clustering and a generalized regression approach. Cancer Res 27:209–220PubMedGoogle Scholar
  37. Mao AA, Kharbuli P (2002) Distribution and status of Nepenthes khasiana Hook. f.—a rare endemic pitcher plant of Meghalaya, India. Phytotaxonomy 2:77–83Google Scholar
  38. Mokkamul P, Chaveerach A, Sudmoon R, Tavee T (2007) Species identification and sex determination of the genus Nepenthes (Nepenthaceae). Pak J Biol Sci 10:561–567. doi: 10.3923/pjbs.2007.561.567 CrossRefPubMedGoogle Scholar
  39. Mukerjee A, Dam DP, Dam N (1984) Pitcher plant—an ornamental climber of Meghalaya. Ind Hort April–June 1:6–18Google Scholar
  40. Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent J (2000) Biodiversity hotspots for conservation priorities. Nature 403:853–858CrossRefPubMedGoogle Scholar
  41. Oostermeijer JGB, Luijten SH, denNijs JCM (2003) Integrating demographic and genetic approaches in plant conservation. Biol Conserv 113:389–398. doi: 10.1016/S0006-3207(03)00127-7 CrossRefGoogle Scholar
  42. Prevost A, Wilkinson MJ (1999) A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theor Appl Genet 98:107–112. doi: 10.1007/s001220051046 CrossRefGoogle Scholar
  43. Rao TA, Shanware PG, Tribedi GN (1969) A note on the pitcher plant habitat in Assam. Ind For 95:611–613Google Scholar
  44. Reynolds EH (1987) Early treatment and prognosis in epilepsy. Epilepsia 28:97–106. doi: 10.1111/j.1528-1157.1987.tb03633.x CrossRefPubMedGoogle Scholar
  45. Sokal RR, Sneath PHA (1963) Principles of Numeric Taxonomy. Freeman, San Francisco, p 359Google Scholar
  46. Soltis PS, Soltis DE (1991) Multiple origins of the allotetraploid Tragopogon mirus (Compositae): rDNA evidence. Syst Bot 16:407–413. doi: 10.2307/2419333 CrossRefGoogle Scholar
  47. Tamaki I, Seteku S, Tomaru N (2008) Genetic variation and differentiation in populations of a threatened tree, Magnolia stellata: factors influencing the level of within-population genetic variation. Heredity 100:415–423. doi: 10.1038/sj.hdy.6801097 CrossRefPubMedGoogle Scholar
  48. Thomson RH (1987) Naturally occurring quinones III. Chapman & Hall, New York Google Scholar
  49. Williams CG, Hamrick JL (1996) Elite populations for conifer breeding and gene conservation. Can J Res 26:453–461. doi: 10.1139/x26-051 CrossRefGoogle Scholar
  50. Young YA, Boyel T, Brown T (1996) The population genetic consequences of habitat fragmentation for plants. Trends Ecol Evol 11:413–418. doi: 10.1016/0169-5347(96)10045-8 CrossRefGoogle Scholar
  51. Zietkiewicz E, Rafalski A, Labuda D (1994) Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics 20:176–183. doi: 10.1006/geno.1994.1151 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • B. S. Bhau
    • 1
    Email author
  • K. Medhi
    • 1
  • T. Sarkar
    • 1
  • S. P. Saikia
    • 1
  1. 1.Plant Genomics laboratory, Medicinal Aromatic and Economic Plants (MAEP) DivisionNorth-East Institute of Science and Technology (NEIST)-Jorhat (Formerly RRL)JorhatIndia

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